晚期糖基化终产物对内皮细胞Connexin40基因表达的影响

目的研究晚期糖基化终产物（advanced glycation end products，AGEs）对体外培养的人脐静脉内皮细胞缝隙连接蛋白Connexin40基因表达的影响。方法D-葡萄糖孵育牛血清白蛋白制备糖基化牛血清白蛋白作为晚期糖基化终产物。体外常规培养的人脐静脉内皮细胞分为4组，分别为对照组、100mg／LAGEs干预组、200mg／LAGEs干预组和400mg／LAGEs干预组，干预24h后通过RT—PCR方法检测各组Connexin40基因mRNA的表达有无差异；通过免疫细胞荧光方法检测对照组和200mg／LAGEs干预组之间Connexin40基因蛋白的表达水平有无差异。结果不同浓度（100、200、400mg／L）的AGEs干预24h均能引起人脐静脉内皮细胞Connexin40基因mRNA的表达减少（均P〈0．01）；200mg／L的AGEs干预24h引起人脐静脉内皮细胞Con—nexin40基因的蛋白表达减少（P〈0．01）结论AGEs体外短期干预可引起人脐静脉内皮细胞Connexin40基因的mRNA和蛋白表达水平减少。

氧化低密度脂蛋白对人脐静脉内皮细胞Connexin40表达的影响

目的:研究氧化低密度脂蛋白(OxidizedLDL,Ox-LDL)对体外培养的人脐静脉内皮细胞缝隙连接蛋白(Gap junction protein)Connexin40基因表达的影响。方法:不同浓度Ox-LDL的干预:体外培养的内皮细胞分为4组:对照组,50mg/L,100mg/L,200mg/LOx-LDL干预组,干预24小时后通过RT-PCR方法检测各组Connexin40基因mRNA的表达有无差异;不同时间Ox-LDL干预的内皮细胞分为对照组,50mg/L的Ox-LDL干预6小时、12小时、24小时、48小时和72小时组共6组,通过RT-PCR方法检测各组Connexin40 mRNA的表达有无差异;蛋白表达水平的检测:通过免疫细胞化学方法检测对照组和100mg/LOx-LDL干预组之间Connexin40蛋白的表达水平有无差异。结果:不同浓度(50mg/L,100mg/L,200mg/L)的Ox-LDL干预24小时能引起内皮细胞Connexin40mRNA的表达增加(P<0.01);50mg/L的Ox-LDL干预不同时间后Connexin40mRNA表达增加,以6小时和12小时最明显(P<0.01);100mg/L的Ox-LDL干预24小时内皮细胞Connexin40蛋白表达增加(P<0.01)。结论:Ox-LDL短期干预可引起人脐静脉内皮细胞Connexin40基因的mRNA和蛋白表达水平增加。

缝隙连接蛋白40和心房颤动

随着对心房颤动机制越来越深入的了解,缝隙连接蛋白在其中扮演的作用日益受到重视。缝隙连接蛋白中的连接蛋白40在心房颤动过程中数量、分布、及分子结构都发生了变化。连接蛋白40基因突变及多态性增加了心房颤动的易患性,尽管连接蛋白40在心房颤动发生和维持中的具体机制尚未完全阐明,但一些针对连接蛋白的药物应用于心律失常有较好的效果。现就连接蛋白40与心房颤动的关系进行综述。

血管紧张素Ⅱ对大鼠心房Cx40重构的影响及贝那普利的干预作用

目的采用大鼠肾上腹主动脉部分结扎的模型,观察血管紧张素Ⅱ(AngⅡ)对心房缝隙连接蛋白(connexin,Cx)40重构的影响及贝那普利的干预效果。方法将30只雄性Wistar大鼠随机数字表法分为对照组(C组)、腹主动脉结扎组(A组)、结扎+贝那普利组(B组)。喂养8周后处死,用放射免疫法测血浆及心肌组织AngⅡ含量,用免疫组化方法检测大鼠心房Cx40的分布特征,半定量分析Cx40的含量。结果与C组相比,A组血浆、心肌AngⅡ含量均明显升高(P<0.01),而B组则无明显变化(P>0.05)。A组大鼠心房Cx40含量减少(P<0.01),并向侧边分布增加;而B组Cx40含量较A组增加(P<0.01),且分布不均一程度减轻。结论AngⅡ升高可导致心房Cx40重新分布和表达减少;贝那普利通过降低AngⅡ水平,可有效减轻心房Cx40的重构。

Connexin 40-formed GJIC increases the phototoxicity of photodynamic therapy through ROS-and calcium-mediated pathways

OBJECTIVE To explore the effect of connexin(Cx)40-formed gap junctional intercellular communication(GJIC)on Photofrin-photodynamic therapy(PDT)phototoxicity in Cx40-transfected He La cells and its potential mechanisms.METHODS He La cell line stably transfected to express Cx40 was seeded at high and low cell density,respectively,to assess in vitro photosensitivity using CCK8 assay.Western blot assay was performed to detect the expression of Cx40.The intracellular ROS and Ca^(2+) concentrations were determined using flow cytometer.4-HNE and ceramide were measured using ELISA assay.RESULTS Cx40-composed GJ formation at high density enhances the phototoxicity of PhotofrinPDT.When the Cx40 is not expressed or Cx40 channels are blocked,the phototoxicity in high-density cultures substantially reduces,indicating that the enhanced PDT phototoxicity at high density is mediated by Cx40-composed GJIC.The GJIC-mediated increase in PDT phototoxicity was associated with ROS and calcium-mediated stress signaling pathways.CONCLUSION The work uniquely presents the ability of Cx40-composed GJIC to enhance the sensitivity of malignant cells to PDT,and indicates that maintenance or increase of Cx40-formed GJIC may be a profitable strategy towards the enhancement of PDT therapeutic efficiency.

Effect of cAMP and cGMP on Connexin43 Expression in Isolated Human and Bovine Ciliary Epithelium

The aim of the study was to assess the distribution of connexin43 (Cx43) and connexin40 (Cx40) in human and bovine ciliary bodies. The effect of the second messengers cAMP and cyclic cGMP on Cx43 protein expression was also investigated. Enucleated human eyes (remnant after corneal transplantation) and bovine eyes were used. Tissue preparations of the anterior segments of the eyes have proceeded for immunohistochemical staining with polyclonal antibodies of Cx43 and Cx40. Isolated ciliary bodies of human and bovine eyes were incubated with cAMP analog 8-Bromo-cAMP or the cGMP analog 8-Bromo-cGMP, the expression of Cx43 protein in the tissues was then assessed by Western blot assay. Both in human and bovine ciliary bodies, strong immunoreactivity of Cx43, but not Cx40, was observed predominantly in the apical cytoplasmic portions of the pigment ciliary epithelial cells and non-pigmented ciliary epithelial cells. In human ciliary body both cAMP and cGMP up-regulated Cx43 expression, while in the bovine ciliary body, cGMP increased Cx43 expression but cAMP decreased it. Cx43 is the major component of human and bovine gap junctions in the ciliary epithelium. The regulation on the Cx43 expression by cAMP and cGMP in human and bovine ciliary bodies suggests the possibly different roles of these signal messengers in the intracellular communication.

瓣膜病慢性心房颤动心房肌缝隙连接CX43和CX40的重构

目的 探讨人类瓣膜病慢性心房颤动 (房颤 )心房肌中缝隙连接通道蛋白Connexin 4 3(CX4 3)和Connexin 4 0 (CX4 0 )的重构及其与房颤的关系。方法 (1)取 11例风湿性心脏瓣膜病 (风心病 )患者的右心耳心肌 (房颤 8例 ,窦性心律 3例 ) ,以Northernblot印迹法比较窦性心律和房颤者CX4 3和CX4 0mRNA表达量的变化 ;(2 )另取 2 0例风心病患者右心耳心肌 (窦性心律 8例 ,房颤 12例 ,房颤持续时间从 3个月至 15年 )制成冰冻切片 ,在激光共聚焦显微镜 (confocallaserscanningmi croscopy)下分别观察窦性心律和房颤心房肌CX4 3和CX4 0蛋白的荧光斑面积、空间分布及形态变化。结果 (1)窦性心律和房颤CX4 3和CX4 0mRNA表达量差异无显著性 ;(2 )CX4 3和CX4 0荧光斑面积在窦性心律和房颤时差异也无显著性 ,但CX4 0在心肌纵切面上的数目明显多于横切面 (P <0 0 5 ) ,而CX4 3数目无变化。提示房颤时CX4 0通道在心肌空间上的分布发生了显著改变 ,即端 端连接大于侧 侧连接。结论 人类风心病慢性房颤时缝隙连接蛋白CX4 3和CX4 0的mRNA表达量和蛋白量均未发生显著改变 ,但CX4 0蛋白的空间分布发生了显著变化 ,端 端连接明显增多 ,侧 侧连接显著减少 ,提示房颤时CX4 0通道发生了空间排列上的重构 。